title: Near-ultraviolet circular dichroic activity of apomyoglobin: resolution of the individual tryptophanyl contributions by site-directed mutagenesis.
abstract: The individual tryptophanyl contributions to the near-ultraviolet circular dichroic activity of apomyoglobin in its native conformation have been resolved by studying recombinant proteins with single tryptophanyl substitutions. Site-directed mutagenesis of sperm whale apomyoglobin was performed in order to obtain proteins containing only Trp A-5 or Trp A-12. These amino acid substitutions have very little effect on the overall globin fold as indicated by comparing the spectroscopic properties of the mutants with those of the wild type protein. The circular dichroism spectra of the two apomyoglobin mutants in the near ultraviolet were found to be significantly different, both indole residues having significant activity but of opposite sign. In particular, Trp A-5 shows the presence of a main positive peak centered near 294-295 nm with a marked shoulder at 285 nm, ascribed to the 1LB transition. The spectrum of the mutant protein containing only Trp A-12 shows a large negative contribution with a minimum near 283 nm and a marked shoulder at 293 nm. The broadness of the negative contribution exhibited by Trp A-12 suggests that it may originate mainly from the 1LA transition.
authors.affiliation: Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Sirangelo
authors.forename: E
authors.initials: E
authors.lastname: Bismuto
authors.forename: S
authors.initials: S
authors.lastname: Tavassi
authors.forename: G
authors.initials: G
authors.lastname: Irace
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry*; D014907:Whales
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin; D014364:Tryptophan